The present invention relates to the area of cancer therapeutics. More particularly, the present invention relates to fragments of Fen1 that interact with PCNA, and the use of such fragments or mimetics of Fen1 in methods of screening for compounds useful in treating disorders in which PCNA is implicated.
Maintenance of genomic integrity within the cell requires a co-ordination between cell-cycle regulated DNA replication, and DNA repair. In the presence of damaged DNA, proliferating cells must cease DNA replication, so that lesions do not become fixed, and repair all damage before replication can recommence. Therefore, the co-ordination of these two processes is critical to avoid mutation and genomic instability. One protein known to be involved in both in DNA replication and in nucleotide excision repair is proliferating cell nuclear antigen (PCNA).
In solution, PCNA from Saccharomyces cerevisiae is thought to exist as a trimer. Each monomer has two structurally similar domains separated by a central loop, and so the trimer shows overall six-fold symmetry, as determined by X-ray crystallographic analysis (Kong et al., 1992; Krishna et al., 1994). Despite variation at the amino acid level, human PCNA is thought to be highly homologous at the structural level to budding yeast PCNA (Krishna et al., 1994). These structural studies have shown that trimeric PCNA forms a toroidal structure around DNA, confirming earlier biochemical studies that suggested that PCNA acts as a sliding clamp around double stranded DNA (reviewed by Kuriyan and O""Donnell, 1993), holding the DNA replication machinery onto its template and thereby greatly enhancing its processivity (Bravo et al., 1987; Prelich et al., 1987b). PCNA is localised to sites of DNA synthesis within the nucleus (eg Bravo and MacDonald-Bravo, 1985), and is required to reconstitute SV40 DNA replication in vitro from purified proteins (Prelich et al., 1987a), clearly demonstrating a requirement for the protein in DNA replication. Similarly, the Schizosaccharomyces pombe PCNA gene pcn1 is essential, with cells showing a phenotype characteristic of a defect in DNA replication when pcn1 is deleted (Waseem et al., 1992). In addition to its replication role, PCNA is also required for nucleotide excision repair in cell-free systems (Shivji et al., 1992). However, the way in which PCNA carries out these two separate roles is as yet unclear.
The present invention is based on the finding that a human protein Fen1 interacts with PCNA. This was shown using a yeast two hybrid screen for proteins encoded by a human cDNA library that interact with human PCNA in a cellular environment.
Fen1 has previously been described as a structure-specific endonuclease (Harrington and Lieber, 1994a) with 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity (Robins et al., 1994) that shares homology with putative nucleotide excision repair factors including human xeroderma pigmentosum complementation G group protein (Harbraken et al., 1994; O""Donovan et al., 1994), S. pombe rad 2 and rad13 (Carr et al., 1993; Murray et al., 1994), and S. cerevisiae RAD27/YKL510 and RAD2 (Jacquier et al., 1992; Siede and Friedberg, 1992). The same protein has, however, been identified as an essential DNA replication factor MF1 (Waga et al., 1994a).
The present invention further relates to the characterisation of the interaction between Fen1 and PCNA at the amino acid level by mapping of the mutual binding sites of each protein. This revealed that p21Cip1 (also known as p21WAF1 or Sdi1), the cyclin-kinase inhibitor that also blocks PCNA""s function in DNA replication (Flores et al., 1994; Waga et al., 1994b; Warbrick et al., 1995) but not repair (Li et al., 1994; Shivji et al., 1994), binds to the same site on PCNA as does Fen1. The regions of Fen1 and p21Cip1 that interact with PCNA are shown to be homologous, and p21Cip1 peptides are found to compete with Fen1 for binding to PCNA.
The finding that p21Cip1, or fragments thereof, compete with Fen1 for PCNA, in particular the region of p21Cip1 identified in our copending application number PCT/GB95/02583 as being responsible for PCNA binding, leads to the possibility of using Fen1 in the screening of mimetics for p21Cip1, in particular those which may block or inhibit cellular DNA replication.
Accordingly, in one aspect, the present invention provides a substance which has the property of binding to PCNA, said substance comprising:
(i) a fragment of the Fen1 protein containing a peptide of 89 amino acids from the C-terminal region or an active portion thereof; or,
(ii) a fragment of the Fen1 protein containing the sequence motif QGRLDxFF (SEQ ID NO:1); or
(iii) a functional mimetic of said protein fragments.
We have found that xe2x80x9cxxe2x80x9d may be S, D or G, but probably other amino acids will be tolerated as well.
In the present invention, xe2x80x9can active portionxe2x80x9d means a peptide which is less than said full length Fen1 amino acid sequence, but which retains the property of binding to PCNA.
In the present invention, xe2x80x9cfunctional mimeticxe2x80x9d means a substance which may not be a peptide at all, but which has the property of binding to PCNA, excluding the p21Cip1 fragments disclosed in our earlier application.
In a further aspect, the present invention provides assays using a binding agent which is a fragment or mimetic of Fen1 as described above. In particular, the present invention provides a method of screening for Fen1 or p21Cip1 mimetics comprising exposing Fen1 or a fragment or mimetic thereof which binds PCNA (herein referred to as xe2x80x9cthe Fen1 componentxe2x80x9d) and a candidate mimetic to PCNA or an active fragment thereof (herein referred to as xe2x80x9cthe PCNA componentxe2x80x9d), so that the candidate mimetic and the Fen1 component compete to bind the PCNA component, and detecting the extent of binding of the PCNA component to the candidate mimetic and/or the Fen1 component. Candidate mimetics which are found to bind to PCNA can then be further screened for biological activity, especially inhibition of DNA synthesis or inhibition of (tumour) cell growth.
Conveniently, the screening method can be carried out by immobilising the fragment or mimetic of Fen1 on a solid support, and exposing the immobilised Fen1 component to PCNA and various concentrations of the candidate mimetic. The extent of PCNA binding to immobilised Fen1 can be measured using an antibody which detects PCNA. Alternatively, interaction of radiolabelled PCNA with immobilised Fen1 component in the presence of candidate mimetic can be measured in a scintillation proximity assay. Other assay formats and screening techniques using Fen1 fragments or mimetics can be readily determined by the skilled person and used to screen candidate mimetics.
In a further aspect, the present invention includes mimetics obtained by using the above screening method.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a xe2x80x9cleadxe2x80x9d compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides may be unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in conferring the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, eg by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its xe2x80x9cpharmacophorexe2x80x9d.
Once the pharmacophore has been found, its structure is modelled according to its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques such as NMR, X-ray diffraction data, etc. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and has the desired degradation profile in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vitro, in vivo or clinical testing.
The final mimetic may have clinical utility, or it may be useful as a drug in a laboratory setting. In clinical uses, the desired degradation profile may be one in which degradation is minimised, but there are situations where an appropriate rate of degradation is not only desired, but is actually important. For example, the p21Cip1 mimetic could be used as a short half-life adjunct to arrest cell proliferation transiently in normal cells while administering genotoxic compounds to cells that override the p21Cip1 arrest, for example cancer cells which contain abnormally high levels of PCNA.
Thus, in another aspect, the present invention provides the class of peptides, and also mimetics obtained as described above, based on the PCNA-binding region of Fen1. These compounds, and especially the mimetics, may be useful in the preparation of pharmaceuticals for treating conditions in which PCNA is implicated, including hyperproliferative diseases, such as cancer and psoriasis.
Therapeutic applications of the present invention include the administration of the various peptides or mimetics mentioned above. Various methods of administration of the therapeutic agent can be used, following known formulations and procedures. Dosages can be determined by routine experimentation. The administration may be systemic or targeted, the latter employing direct (eg topical) application of the therapeutic agent to the target cells or the use of targeting systems such as cell type specific antibodies or ligands. Targeting is generally preferable since it minimises or localises any side effects; and may be particularly important for example if the agent is unacceptably toxic when administered systemically, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
Instead of administering these agents directly, they could be produced in the target cells by expression from an encoding gene introduced into the cells, eg in a viral vector (a variant of the VDEPT techniquexe2x80x94see below). The vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are switched on more or less selectively within the target cells.
Alternatively, the agent could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. This type of approach is sometimes known as ADEPT or VDEPT; the former involving targeting the activating agent to the cells by conjugation to a cell type specific antibody, while the latter involves producing the activating agent, eg an enzyme, by expression from encoding DNA in a viral vector (see for example, EP-A-415731 and WO 90/07936).
In a further aspect, the present invention identifies the region of PCNA involved in binding to Fen1. Thus, the present invention provides a substance which has the property of binding to Fen1, said substance comprising a fragment of PCNA lying between amino acids 100-150, or an active portion thereof; or a functional mimetic of said protein fragments.
If fragments of PCNA in this region retain the ability to bind to p21Cip1 or Fen1, it may be possible to use these: (i) to regulate p21Cip1 dosage, (ii) to screen for Fen1 or p21Cip1 mimetics and in the design of substances to bind to this site in PCNA; (iii) to induce proliferation in hypoproliferative cells, for example to reactivate senescent cells.